Stem Cells
Stem cells are undifferentiated primal cells that are capable of differentiating into cells of various other types. Because of their ability to form various types of differentiated cells, stem cells function to replenish other cells throughout the life of the organism.
Research efforts are currently aimed at developing methods wherein stem cells are used to repair damaged tissues or to grow replacement organs or body parts. In order for stem cells to be routinely useable for such therapeutic applications, it will be necessary to develop techniques for efficiently and reliably identifying and isolating stem cells from populations of other differentiated cells.
Stem Cell Identification and Isolation
Techniques to isolate adult and embryonic stem cells for clinical and experimental use are in development by many workers. However, although much effort has been expended in searching for stem cell probes, currently there still exists no reliably specific marker for stem cells of any type. Additionally, many of the markers that have been proposed and studied are invasive to the cell or cell surface and may alter or even kill the cell.
One of the most accepted properties of stem cells is their ability to slowly cycle and continuously replenish cell populations. We hypothesized and found that such slow cycling cells can be characterized by slower intrinsic metabolism. As such, these slow cycling populations are detectable via metabolic monitoring. We have developed a novel fluorescence spectroscopic technique and device to separate live, unlabeled stem cells from further differentiated stages of development based on this simple finding.
Currently there exists no reliable, non-invasive technique to specifically detect or screen for the presence of stem cells, or to reliably isolate stem cells from a matrix that contains stem cells along with other types of cells. Development of such a technique, especially a generalizable one, would not only represent a unique stem cell detection and separation method/device, but would also provide a viable alternative to the currently popular search for lineage-specific markers.
Mitochondria
Mitochondria are elongate or rod shaped, membrane-enclosed, organelles located within cells of the body. Mitochondria function as the major source of a cell's energy by oxidizing the products of carbohydrate and lipid metabolism. Mitochondria are located outside the nucleus of the cell but contain some independent DNA and may be reproduced as needed by the cell within which they reside. Damage to mitochondria can result in muscular weakness and fatigue and, in some cases, may lead to life-threatening conditions, such as lactic acidosis. Nucleoside analogs may cause mitochondrial toxicity.
In normal healthy cells, mitochondria are diffusely distributed throughout the cytoplasm. Abnormal clustering of mitochondria in the perinuclear region of the cell can be indicative of changes in the metabolic state of the cell. Existing literature supports two mechanisms to explain how mitochondrial redistribution occurs. Firstly, these organelles may simply migrate via a complex microtubular network from the perinuclear region to the cell periphery. In cells transfected with a mutant member of dynamin, a family of membrane transport proteins, perinuclear aggregates of mitochondria were demonstrated by electron microscopy, whereas cells with normal tubular projections exhibited diffuse mitochondrial arrangement. Impairment of kinesin-mediated transport by tumor necrosis factor-related apoptosis inducing ligand (TRAIL) also leads to these changes in mitochondrial distribution. Furthermore, treatment with microtubule-active drugs (taxol, nocodazole and colchicine) results in perinuclear clustering of mitochondria. The second mechanism involves the presence of two populations of mitochondria, perinuclear and peripheral, as observed by confocal fluorescence microscopy of mitochondria labeled with two potentiometric probes, rhodamine 123 and dimethylaminostyryl-methylpyridiniumiodine, with each population exhibiting different levels of activity and morphology depending on cell type. Cultured cancer cells have exhibited increased perinuclear fluorescence in strains sensitive to chemotherapeutic drugs and increased peripheral activity and consequently fluorescence, in the mitochondria of resistant strains.
There exists a need for the development of new techniques for assessing changes or abnormalities of mitochondrial distribution and structure in living cells without the need for killing the cell or destroying the tissue within which the cell is located.
Reduction-oxidation (Redox) Fluorometry
Redox Fluorometry is an optical spectroscopic technique wherein autofluorescence is measured from reduced pyridine nucleotides (PN) and oxidized flavoproteins (Fp). The ratio of PN to Fp (PN/Fp ratio) is then calculated. This PN/Fp ratio may be used as an indicator of tissue metabolic rate.
In the performance of redox fluorometry, the amount of PN (i.e., NADH and NADPH) may be estimated by detecting fluorescence emission in the region of 450 nm after excitation at 366 nm. This estimate includes both cytoplasmic and mitochondrial NADH and NADPH, with greater quantum yield from the mitochondrial bound species. The amount of Fp (i.e., lipoamide dehydrogenase (LipDH) and electron transfer flavoprotein (ETF)) is then estimated by detection of fluorescence in the region of 540 nm after excitation at 460 nm. This measures cellular levels of the flavoproteins which exist mostly as co-factors for enzymes involved in redox reactions. The ratio of these fluorescence measurements, which minimizes interfering factors such as absorption of excitation and emission light by other intrinsic chromophores, light scattering, and variations in mitochondrial density and flavoprotein concentration, has previously been proposed as a non-invasive measure of the organ cellular metabolic state.
Initially, most redox fluorometry methods were performed using one-photon (1P) excitation at near-UV and visible wavelengths for NAD(P)H and FP fluorescence. However, the use of 1P-redox fluorometry to determine PN/Fp ratios of cells in situ was found to be problematic due to photobleaching of intrinsic fluorophores and other light-induced damage as well as light scattering and absorption in turbid cell and tissue environments. These problems with 1P-redox fluorometry were largely overcome by the use of multiphoton microscopy coupled with near-infrared (NIR) excitation. MPM offers several advantages including a) little or no photobleaching while out of focus, b) three-dimensional resolution, c) less light scattering and photodamage and the ability to determine PN/Fp ratio in tissue planes that are below the surface of an organ or tissue mass. Thus, two-photon (2P) NAD(P)H fluorescence has become a preferred method for performing redox fluorometry of in vivo tissues and some other applications. More recently, the development of two-photon (2P) femtosecond laser excitation and scanning confocal microscopy has enabled the three-dimensional mapping of cellular metabolic oxidation/reduction states in situ with high resolution.
Redox fluorometry has also been applied to the detection of cells with deregulated proliferative potential. Using this non-invasive spectroscopic technique, normal and transformed fibroblasts have been separated, as have proliferating and non-proliferating epithelial cells. More recently others have discovered that intracellular redox state appears to be a necessary and sufficient modulator of the balance between self-renewal and differentiation in dividing optic nerve oligodendrocyte-type-2 astrocyte progenitor cells. That is, the intracellular redox state of freshly isolated progenitors allows prospective isolation of cells with different self-renewal characteristics.
The non-invasive microscopic technique of redox fluorometry, which is based upon stimulated auto fluorescence detection, has been historically suggested as a viable clinical measure of the cellular metabolic state. More recently, redox fluorometry has also been demonstrated to be able to differentiate between self-renewing and differentiating cells.